Hydrogen sulfide (H2S) is a colorless gasotransmitter (gaseous signaling molecule) that plays a vital role in numerous cellular functions within the human body. For instance, over the past decade, the role of H2S beyond a toxicant and environmental pollutant has evolved to encompass several biochemical functions that are important in various physiological and pathological responses such as cardiovascular (dys)function, neurological (dys)function, gastrointestinal (dys)function, immune (dys)function, and several other molecular and cell biology responses.
For instance, H2S has been discovered to have significant potential to contribute to the detection and treatment of cardiovascular disease, including atherosclerosis and peripheral arterial disease. Because of decreased oxidative modification of low-density lipoprotein (LDL), H2S has also been shown to play a significant role in atherosclerosis by having noted effects on monocyte recruitment, transformation into tissue macrophages, and foam cell formation. Further, H2S has been shown to inhibit hypochlorite and hemin-mediated atherogenic modification of LDL. Plasma H2S levels have also been shown to be lower in atherosclerotic plaque, and treatment with sodium hydrosulfide (NaHS) decreases both aortic plaque and intercellular adhesion molecule-1 (ICAM-1). In addition, H2S down regulates the expression of monocyte chemoattractant protein-1, a CC chemokine that binds to the C—C chemokine receptor type 2 (CCR2) and recruits monocytes into the subendothelial layer to form atherosclerotic plaque. Another critical role of H2S in the pathogenesis of atherosclerosis is the effect of inducing apoptosis on vascular smooth muscle cells, which generates atherosclerotic plaque. Hydrogen sulfide, administered as NaHS, decreases the proliferation of vascular smooth muscle cell via a mitogen-activated protein kinase (MAPK) pathway in a dose-dependent fashion in rat models. Additional work with a rat model reveals that H2S reduced vascular calcification. Additionally, recent studies have shown H2S to have a direct relationship with nitrogen monoxide and carbon monoxide in peripheral arterial disease (PAD) identification.
Hydrogen sulfide arises from multiple biological sources and tissues (e.g. bacteria and organ-specific production). Endogenous biological H2S production primarily originates from cysteine metabolism through the activity of cystathionine β-synthase and cystathione γ-lyase or through 3-mercaptopyruvate metabolism by 3-mercaptosulfurtransferase. H2S can come from redox-dependent metabolism of polysulfides involving glutathione or other small molecular weight thiol modifiers. Lastly, H2S also arises from different environmental sources that affect humans such as petroleum production and exploration, food and beverage processing, waste disposal and sewage treatment, agriculture and farming, and bacterial contamination and function.
Hydrogen sulfide chemistry is complex and plays several roles in modulating protein thiol function. It affects numerous biological responses involving signal transduction responses, mitochondrial respiration, gene expression, and cell survival/viability. At a physiological pH of ˜7.2-7.4, H2S predominantly (˜80%) exists in its anion HS− form with a smaller amount in the gaseous H2S form (˜20%). This is due to pKa regulation of H2S forms in aqueous solutions as illustrated in the following equation:pKa1=7.04 pKa2≥13H2S⇄HS−⇄S2−Due to the different pKa's, the ionic distribution is easily manipulated and, in turn, its distribution controlled in either aqueous or gas phases.
Hydrogen sulfide is very reactive within biological or environmental systems, resulting in sulfide equivalents being present in three different volatile sulfur pools as shown in FIG. 1. These three pools—free H2S, acid labile H2S, and sulfane sulfur species—are important in regulating the amount of bioavailable sulfur. Free hydrogen sulfide is found dissolved in plasma and other tissue fluids. At mammalian body conditions (i.e., pH 7.4 and temperature of 37° C.), 18.5% of free hydrogen sulfide exists as H2S gas, and the remainder is almost all hydrosulfide anion (HS−) with a negligible contribution of S2−. Sulfane sulfur refers to divalent sulfur atoms bound to another sulfur, though they may bear an ionizable hydrogen at some pH values. Examples of these bound sulfurs include thiosulfate S2O32−, persulfides R—S—SH, thiosulfaonates R—S(O)—S—R′, polysulfides R—Sn—R, polythionates SnO62−, and elemental sulfur S0. Acid labile sulfide, the other major bioavailable pool, consists of sulfur present in iron-sulfur clusters contained in iron-sulfur proteins (non-heme), which are ubiquitous in living organisms, and include a variety of proteins and enzymes, including without limitation, rubredoxins, ferredoxins, aconitase, and succinate dehydrogenase. The acid labile sulfides readily liberate free H2S in acid conditions (pH<5.4), and the process of acid liberation may also release hydrogen sulfide from persulfides, which have traditionally been classified as sulfane sulfur. This acid labile sulfur pool has been postulated to be a reversible sulfide sink and may be an important storage pool that regulates the amount of bioavailable free hydrogen sulfide.
H2S equivalents are readily mobilized from these pools based on changes in pH, O2 concentration, and oxidative/reductive chemistry that affect biological and biochemical responses. Thus, detection of H2S availability from these distinct pools is important for clinical pathophysiology diagnosis, environmental source identification, and any other organic or inorganic chemistry uses.
Unfortunately, a significant barrier to the study of hydrogen sulfide's role in human health and disease has been the lack of precise methodology and testing means for the accurate and reproducible measurement of hydrogen sulfide both in vivo and in vitro. A variety of methods to measure free H2S have been employed, but with divergent results. These methods include a spectrophotometric derivatization method resulting in methylene blue formation, variations of this methylene blue method using high performance liquid chromatography, sulfide ion-selective electrodes, polarographic sensors, gas chromatography, and high-performance liquid chromatography (HPLC) in conjunction with fluorimetric based methods using monobromobimane (MBB) to derivatize free H2S. The complexity of analytical H2S measurement, especially in living organisms, reflects the fact that hydrogen sulfide is a reactive gas and exists in the organism in the three different volatile sulfur pools shown in FIG. 1. Due to a lack of reliable, accurate analytical detection methods available to quantify H2S and its various forms, there is great disagreement regarding precise amounts and sources of H2S metabolism in biological and biochemical settings. Therefore, there is a great need for an apparatus and associated methodology that can be used to accurately and conveniently measure H2S in its various bioavailable forms.